In response to the exponential increase in the emergence and reemergence of international disease threats and other health risks, 194 countries across the globe agreed to implement the International Health Regulations (2005) (IHR). This international law was enacted on 15 June 2007. The stated purpose and scope of the IHR – “to prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade” − requires States to strengthen core surveillance and response capacities at the primary, intermediate, and national level. An important component of IHR implementation is the national monitoring and evaluation of each country’s capacities to detect, assess, and report potential public health emergencies of global concern. The US has recognized that assessments of preparedness and response capacities using the Joint External Evaluation Tool (JEE) and subsequent development of the JEE national action plans are critical priorities for countries to enhance their national health security. The US Government has prioritized the finalization and public release of the JEE National Action Plan as part of this commitment. Increasing global risks of nuclear and radiological accidents or attacks continue to challenge national capacities to achieve the global social norms against nuclear proliferation while maintaining global security. This has driven renewed research interest in developing medical countermeasures for clinically significant ionizing radiation exposure. The availability of safe and effective countermeasures against these threats currently represents a significant unmet medical need. To expedite CBRN (chemical, biological, radiological, and nuclear) countermeasure development, the FDA has implemented the “Animal Rule” which applies to the development and testing of drugs and biologicals to reduce or prevent life-threatening conditions caused by exposure to lethal or permanently disabling CBRN agents, where human efficacy trials are not feasible or ethical. Several drugs for CB indications and two agents for ARS, Neupogen and Neulasta, have been repurposed and approved by the US FDA following the Animal Rule. Repurposing is vital for making drugs available for CBRN threats. Drugs are commonly repurposed for new indications not originally envisioned. By repurposing a drug, one can make it available via FDA approval much faster, but this process also involves intellectual property issues as corporations are hesitant to risk exposing their blockbuster drugs to additional scientific scrutiny. As stated above, both radiation countermeasures approved for hematopoietic ARS following the Animal Rule (Neupogen and Neulasta) are repurposed agents. The emergency use authorization (EUA) is a legal means for the FDA to approve new drugs or new indications for previously approved drugs for use during a declared emergency and is a valid way to expedite drug development. The EUA authority allows the FDA to strengthen the nation’s public health protections against CBRN threats by facilitating the availability and use of countermeasures needed during public health emergencies. In brief, we need to make concerted efforts in global health security through the development of radiation countermeasures.

Exposures to ionizing radiation, whether they are intended or unintended, are currently an undeniable reality and carry potentially catastrophic health consequences. Therefore, medical preparedness and countermeasures are critical security issues, not only for the individual, but for the nation as a whole. Acute radiation exposure induces apoptosis of hematopoietic, digestive, cutaneous, cardiovascular, and nervous system tissues; extensive apoptosis ultimately leads to acute radiation syndrome (ARS). Significant scientific advances have been made over the last six decades toward the development of a safe, non-toxic and effective radiation countermeasures for ARS. Several candidate drugs for ARS have been identified which have low toxicity and significant radio-protective and radio-mitigative efficacy. Ongoing studies in our laboratory suggest that a few of these agentsareprogressingwellalongthe US FDA approvalpathway. Since exposing healthy human volunteers to injurious levels of radiation is unethical, development and approval of new radiation countermeasures for ARS are therefore presently based on animal studies and Phase I safety/pharmacokinetic study in healthy volunteers. The Animal Rule that underlies the U.S. Food and Drug Administration (FDA) approval pathway requires a sound understanding of the mechanisms of injury, drug efficacy, and efficacy biomarkers. Gamma-tocotrienol (GT3) is one of the eight isomers (tocols) of vitamin E and appears to be one of the most promising radio-protective tocols. GT3 has been demonstrated to increase survival in rodents, through ameliorating the radiation-induced injuries of the hematopoietic and gastrointestinal systems. When administered 24 h prior to irradiation, GT3 significantly protected irradiated mice and induced high levels of G-CSF. Injection of a G-CSF neutralizing antibody to GT3-treated mice resulted in complete neutralization of G-CSF and abrogation of its radio-protective efficacy in the murine model. Studies conducted in nonhuman primates demonstrated that the GT3 treatment significantly decreased the duration and severity of neutropenia and thrombocytopenia in irradiated NHPs. GT3 administered in one dose was comparable to multiple doses of G-CSF and two PEGylated G-CSF administrations in combination with supportive care, in terms of improving radiation-induced neutropenia and thrombocytopenia. GT3 modulated several metabolomes, lipidomes, microRNA biomarkers in serum and mitigated radiation-dependent transcriptomic changes within the fronto-limbic circuit in NHPs. In brief, our studies indicate that GT3 is a promising radiation countermeasure and there are several promising biomarkers for its efficacy. Disclaimer: The views expressed do not necessarily represent the Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, the Department of Defense, or the United states.

IL-02 (Abstract No.: 0069)

A Promising Molecule Protecting Biological Tissues against Radiation: An Update on a Decade Research Work

Current study is on a drug, under formulation at INMAS, DRDO, to protect personnel who may be deputed for post accidental clean-up operation in radiation contaminated zone. The formulation prepared from a single compound, coded as G-005M, has been extensively studied in our laboratory for its bio-escorting behavior towards cellular entities (DNA, Lipid, and proteins) against lethal radiation. G-005M is able to effectively protect hematopoietic, gastrointestinal tract and respiratory organ systems in lethally irradiated mice. Functionally the compound used in G-005M, reflects high but reversible affinity with beta tubulin, inhibiting microtubule assembly required for mitotic spindle formation inevitable for a cell heading towards its division. Jejunal proteome studies in irradiated mouse model demonstrated appropriate regulation of pro/anti-apoptotic and cytoskeleton proteins by intervention of G-005M. Radiation induced pneumonitis and fibrosis in mouse could also be significantly prohibited by G-005M. Prophylactic administration of G-005M in mice, protected bone marrow stem cells in significant number, which after a brief interval, were able to support desired hematopoietic expansion. G-CSF and IL6 have been significantly regulated by G-005M alone as well with radiation, promoting blood cells for fast proliferation and differentiation. Repeat dosing of the formulation is able to retain irradiated animal survival for longer period. Larger bioavailability, acceptable toxicity profile and 1.33 DMF have motivated us to promote G-005M to the next level of investigation where non-human primates will be evaluated for its translational purpose. Optimistically, G-005M will be soon available against radiation for our rescue teams in specific and civil society in general.

IL-03 (Abstract No.: 0005)

Role of Neutraceutical Compounds from the Herbal Sources in Ameliorating the Radiation Induced Oxidative Damage

Radiation induces numerous deleterious effects at physiological, metabolic and behavioral levels in the living systems. This effect is because of the production of reactive oxygen species, which results in damage to biomolecules. The deleterious effect of the radiation mainly depends on the type of radiation exposure, duration of exposure, dose of radiation exposed and area of radiation exposure. Radio-protectors may be classified as protector, mitigators and sensitizers. A number of chemicals were studied to assess the radio-protective potential. But only amifostine could enter the human trials. So the interest to find an effective radioprotector from an alternative source lead the researchers towards the herbal sources which has been used in the traditional medicine with potent free radical scavenging activity and less toxic at higher dosage.In our laboratory we are working on Nardostachys jatamanasi, Curcumin longa, Caricca Papaya, Cocus neucifera water. To induce oxidative stress Swiss albino mice were irradiated with a single sub lethal dose of 6 Gy whole body electron beam radiation. The levels of oxidative stress were assessed by evaluating the total antioxidant capacity and levels of antioxidant enzymes in the RBC lysate. To assess genotoxicity, comet assay, micronucleus assay and chromosomal aberration assay was carried out. The root extract of Nardostachys jatamanasi and Curcumin longa, Leaf extract of C. papaya and Cocus neucifera water intervention showed reduction in radiation induced oxidative stress in mice model by improving the antioxidant levels and decreasing the genotoxicity levels in mice.

Thrombocytopenia or chronic depletion of platelets in blood could create a life-threatening condition in patients who receive aggressive systemic radiation and chemotherapy. Currently there are no approved agents for the rapid treatment of thrombocytopenia and conditions causing platelet imbalances. We demonstrated that dietary administration of Tulsi (Ocimum sanctum) leaves or subcutaneous administration of Tulsi flavonoid, Orientin flavonoid, results in a significant increase in circulating platelets in a clinically relevant mouse model of partial ablative total body irradiation. No noticeable effects were observed on red blood cells, white blood cells and other hematologic parameters in treated animals indicating the specificity of Tulsi and Orientin towards enhancing platelet formation. The gene expression and immunophenotyping of bone marrow revealed that Orientin stimulates megakaryopoiesis specific transcriptional program. A significant increase in colony formation in bone marrow cells from Orientin pretreated mice, further complemented the effect of Orientin on progenitor cells. The ex-vivo differentiation of irradiated human peripheral blood CD34+ demonstrated stimulatory effect of Orientin on Megakaryocyte Erythrocyte Progenitors (MEP). Thrombocytopenia and platelet imbalances are reported in more than 30% hematological admissions in hospitals, and a significant percentage of these patients need treatment, including platelet transfusion to prevent bleeding. Thrombocytopenia is a major problem in mosquito-borne viral diseases such as dengue fever and dietary administration of Tulsi could be available and economic approach benefiting such patients in the developing world.

IL-05 (Abstract No.: 0065)

An Overview of Medicinally Important Aromatic Plants with Special Reference to Radioprotective Activity: Menthapiperita Linn

Since the prehistoric time, aromatic plants were usually used as natural medicine because of certain remedial as well as pharmacological properties, all over the world. Undoubtedly, developments took place in pharmaceutical industry in recent years and synthesized drugs have been developed for various diseases including cancer. Therefore, interest has been developed to screen and identify the phytochemical with antioxidant activity. Aromatic plants exploited primarily for the essential oil extraction. Essential oils found applications in several industries such as cosmetics, flavoring and fragrance, spices, pesticides and repellent as well as herbal beverages. Several medicinal plants were studied to treat various conventional ailments but only a handful studies are available on aromatic plants particularly in radioprotection. Menthapiperita, commonly called peppermint, is an aromatic, stimulant and carminative and employed for treating nausea, flatulence and vomiting. Mentha extracts have antioxidant properties due to the presence of eugenol, caffeic acid, rosmarinic acid and α-tocopherol. It also contains phenolic acids, flavonoids and s-carvone. Mentha oil was found to afford radioprotection to hematological parameters and phosphatases level in mice. Treatment of M. piperita extract prior exposure to γ radiation in mice has been shown to provide protection in bone marrow cells; it significantly reduced the number of aberrant cells and different chromosomal aberrations in irradiated mice. Also, M. piperita extract pretreatment was efficient in providing protection against hematopoietic injury in bone marrow, intestine and testis in mice. The radioprotective effect of M. piperita was also demonstrated by determining dose modification factor, which was 1.78.

IL-06 (Abstract No.: 0099)

Basic Radiation Biology Research with Relevance to Cancer Radiotherapy

The discoveries of X-rays and radioactivity at the end of 20th century brought revolution in diagnosis and treatment of many diseases including cancer. Early radiobiological studies showed that ionizing radiations were capable to kill the rapidly dividing cells. In fact, physicians were quick to use radiations such as X rays and gamma rays for treatment of cancer patients. However, further studies revealed that radiation does not distinguish between normal and tumor cells thereby imposing a limitation on the dose of radiation for therapy. Most often, due to unacceptable adverse effects of therapeutic radiation on normal tissues, treatment of cancer by radiation has to be discontinued. Based on the observations on radiation cellular effects, it was suggested to employ the strategy of fractionated doses in the clinical settings taking into account the repair of DNA damage induced by radiation. Extensive studies on the mechanism of cellular damage by radiation revealed the intracellular generation of reactive oxygen species (ROS). These cellular radiobiological results allowed the utilization of observed higher oxidative stress status of tumor cells compared to corresponding normal cells in causing selective radiotoxicity in tumor cells. In more recent years, investigations were actively pursued in our laboratory to explore possible higher radiation killing of tumor cells in the presence of some herbal flavonoids. Research results from our Group have shown the promise of several plant-based compounds in combination with radiation to selectively kill the tumor cells while sparing the surrounding normal cells. It is emphasized that future research challenges lie in gaining the deeper insight in the mechanisms of radiation induced damage on normal and tumor cells for developing novel protocols for developing effective treatment of cancer patients.

Department of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal, Karnataka, India

E-mail: rao.satish@manipal.edu

Normal tissue toxicity is the major limitation for achieving the desired therapeutic effect, as well as compromised impact on quality of life after radiotherapy (RT). About 10% of patients after RT developed severe adverse reactions in normal tissues. Therefore, predicting the risk of developing acute reactions before the initiation of RT using cellular and molecular based biomarkers may have its significant clinical implications in personalized medicine. Here we aimed to assess the variation in radioresponse using gamma H2AX foci (DSB marker) in healthy individuals (n=100) and the influence of genetic polymorphisms in DSB repair or related genes to the observed variation. Similar investigation was envisaged the predictive potential of gamma H2AX foci analysis for the risk of acute normal tissue adverse reactions in the breast (n = 132) and head and neck (n=183) cancer patients undergoing RT. Our findings indicated that XRCC6 rs2267437 (P = 0.0340) and LIG4 rs1805388 (P = 0.0250) influenced the DSB repair in healthy individuals. Further, the gamma-H2AX assay and genetic variant in NBN (rs1805794) may have its potential to be developed into a clinically useful predictive biomarker. The insight gained from this extensive investigation may be helpful for the detection of radiosensitive individuals in a healthy population and may have its clinical implications in the prediction of the risk of developing adverse skin reactions in patients undergoing radiotherapy. Acknowledgements: The financial support from DBT (BT/01/COE/06/02/07), Government of India to carry out this research work and Manipal University for providing the research facilities is gratefully acknowledged.

IL-08 (Abstract No.: 0107)

Modulation of Radiation Induced Pulmonary Tissue Response of Pneumonitis by a Oral Selenocystine Formulation in a Mice Model: A Preclinical Study That May Have Potential Clinical Application

We investigated the lung radio-protective efficacy of a novel oral selenocystiene derivative developed in Bhabha Atomic Research Centre (DRUG-X) on irradiated mice. The study was approved by the institutional animal ethics committee. C3H/HeJ (pneumonitis responding) mice received single 18 Gy (0.6 Gy/min), thoracic radiation (RT) and a subset of these were treated with the drug formulation orally (2.5 and 10 mg/kg) thrice weekly 2 hours post-RT and monitored till respiratory distress symptoms appeared. The pneumonitis progression in the irradiated mice was monitored by acquiring CT scans at regular intervals. Further, to study if the drug offered tumour radioprotection, A549 induced tumour xenografts were exposed to thoracic radiation To study the efficacy of oral selenocystine derivative as a lung radioprotector, Bio-equivalence analysis of IP formulation of the drug (2 mg/kg) and oral formulation was done by monitoring the level of selenium by atomic absorption spectroscopy in the lung homogenates prepared at various time points starting from 5 min to 24 h post therapy. Oral formulation had similar pharmacokinetic patterns as IP formulation in the lungs. We found that lung availability (Cmax=0.32 ± 0.05 µg/g) was comparable irrespective of the mode of administration. Lung radioprotection studies were carried out at doses of 2 and 10 mg/kg. Thoracic CT scans performed on biweekly intervals indicated that the pneumonitis started ~ 80 days post-RT and within a week of onset got consolidated in the entire lung. This drug at a dose of 2 mg/kg significantly delayed the progression of pneumonitis (P<0.05) and improved the asymptomatic survival compared to radiation control. ROS activity was also reduced in the lung homogenates of mice treated with Radiation and the experimental radioprotector as compared to radiation alone arm. (p< 0.05). G-CSF levels were reduced when experimental drug was given with radiation suggesting a reduction in acute inflammatory infiltrates when the drug was used along with radiation. Certain pro-inflammatory cytokines like the IL-6 and IL-17 also reduced when the drug was used with radiation signifying that this drug offers radioprotection not only through ROS pathway but also through inflammatory pathway. Surprisingly there was no statistical difference in the TGF-B levels between the radiation alone arm and the radiation + experimental drug arm suggesting that the drug may not be protective against pulmonary fibrosis. The oral formulation this novel selenocystine derivative not only prevents radiation pneumonitis but also reversed the grade of acute pneumonitis in some mice. The above formulation although has protective action against acute inflammatory changes in the lung (pneumonitis) but may not be protective against late pulmonary changes (lung fibrosis).

IL-09 (Abstract No.: 0051)

A Review of the New Herbal Composition (HemoHIM): As a Radioprotective Agent, Immunomodulatory Functional Food, and Supplementary Drug for Cancer Radiotherapy and Chemotherapy

A new herbal composition, HemoHIM, was developed based on protection of self-renewal tissues and hematopoietic system, and restoration of the immune system against radiation damage. HemoHIM was prepared by adding polysaccharide fraction to hot water extract of a new mixture of three edible herbs (Angelica Radix, Cnidium Rhizoma and Paeonia Radix). In vitro, HemoHIM scavenged hydroxyl radicals. In gamma-irradiated mice, HemoHIM enhanced the survival of intestinal crypts, the formation of endogenous spleen colony, and the recovery of white blood cells. Also HemoHIM increased the survival rate of lethally irradiated mice. In 2006, HemoHIM was approved by Korea FDA and commercialized as a immunomodulatory functional food based on the immune modulation effects in cyclophosphamide-induced immune-suppressed mice, aged mice and immune depressed weak-elderly people. Especially, HemoHIM promoted the restoration of the imbalanced Th1/Th2 responses. Recently, for more effective supplementation to cancer therapy, modified HemoHIM (MH-30) was developed by enriching the fat-soluble polyphenols from same herbs as HemoHIM. MH-30 showed higher protective effects on the hematopoietic and self-renewal tissues against radiation injuries than HemoHIM. MH-30 showed the synergistic effect in combination with gamma-ray or anticancer drugs (cisplatin, or paclitaxel) on cancer growth inhibition in cancer cell-implanted mice. Also MH-30 showed the potential to block hepatotoxicity and nephrotoxicity by cisplatin. Based on these results, IND (Investigational New Drug) of MH-30 was approved by K-FDA. MH-30 could be a supplementary drug for enhancing the anti-cancer efficacy and decreasing the adverse effects of cancer radiotherapy and chemotherapy through a clinical study.

IL-10 (Abstract No.: 0108)

Evaluating the Role of Mcl-1 and Its Interacting Partners in Radioresistance of Oral Cancer Cells

Earlier studies from the lab have demonstrated over expression of anti-apoptotic Mcl-1 protein in human oral cancers and its association with radiotherapy resistance and poor outcome, implying it to be a potential therapeutic target in oral cancers. In the present study, we investigated the role of Mcl-1 and its interacting partners, USP9X and TCTP in radio-resistance of oral cancer cells. Expression of these three proteins was induced post radiation treatment, followed by their translocation to the nucleus and co-localization with gamma H2AX, indicating their possible role in DNA damage response. We confirmed the interaction between Mcl-1 and USP9X proteins and demonstrated that USP9X down regulation leads to a significant reduction in Mcl-1 protein levels, primarily due to its increased turnover. Interestingly, the oral cancer patients showing elevated USP9X levels also exhibited high levels of Mcl-1 protein and a significant decrease in disease free survival. Inhibition of USP9X by deubiquitinase inhibitor WP1130, potently induced apoptosis in oral cancer cells associated with a rapid decline of Mcl-1 protein and accumulation of aggresomes. The single agent in vivo efficacy of WP1130 was also demonstrated against oral cancer xenografts. Our studies thus demonstrate an important role of USP9X in stabilizing Mcl-1, thereby contributing to oral cancer radio-resistance. Thus inhibition of Mcl-1and/or their interacting partner holds great promise in the improved management of oral cancer patients.

Despite important technological advances, encephalic radiation-therapy still induces irreversible side effects in cancer patients, justifying the urge to develop new radiation-therapy techniques. Preclinical studies on ultra-high dose rate (Flash) irradiation showed a possibility to efficiently treat the tumors, without inducing drastic side-effects on the normal tissue, by increasing the dose rate over 40 Gy/s. We recently showed that Flash preserves normal brain from radiation-induced memory impairment. In this study, we aimed at investigating the effects of Flash on the normal brain tissue, along with its antitumor effect. In this context, we investigated the cognitive functions several months post whole brain radiation of mice, irradiated at 10 Gy by Flash or Conventional dose rate delivered by an experimental LINAC. Moreover, different glioblastoma models were used to investigate the effect of Flash irradiation.

These models allowed us to confirm the innocuity of Flash irradiation on the normal brain tissue, with an absence of cognitive deficit several months after irradiation at dose rates above 100 Gy/s, coupled with a preservation of neurogenesis and neural stem cells. These results were also observed at the physio pathological level with an absence of astrogliosis and neuroinflammation. Interestingly, we show that Flash irradiation displays an efficient antitumor effect, at least similar to conventional irradiation. Altogether, these results depict the possibility to preserve the normal brain tissue from radiation-induced toxicities by increasing the dose rate over 100 Gy/s. With an antitumor effect equivalent to the conventional dose-rate irradiation, Flash irradiation might become a major contributor to the cancer treatment by radiation therapy.

IL-12 (Abstract No.: 0035)

Cancer Stem Cell Resistance in Bone Marrow

Pranela Rameshwar

Rutgers New Jersey Medical School, Newark, NJ 07103, USA

E-mail: rameshwa@njms.rutgers.edu

Breast cancer dormancy remains an unresolved issue in the field of cancer biology. The cancer cells take advantage of the bone marrow microenvironment to resist treatment and to remain in cellular dormancy for decades. The dormant cells have adapted stem cell properties, referred as the Cancer Stem Cells (CSCs). The CSCs share similar properties with the endogenous hematopoietic stem cells and appear to reside within the same region of the cavity. Thus, there are limits to the use of high dose chemotherapy to target breast cancer cells within the hematopoietic niche for the fear of toxic effects. The seminar will show how non-coding RNA and exosomes mediate the crosstalk between mesenchymal stem cells (MSCs) and the cancer cells to influence the overall response to treatment. Despite a supportive role for MSCs these stem cells can be used as cellular delivery of non-coding RNA. MSCs are currently the preferred stem cells for thousands of clinical trials. The advantage of MSCs over other stem cells is mainly due to the ability to be transplanted as ‘off the shelf’ stem cells, reduced ethical concerns in harvesting the cells, ease in expansion and ‘plasticity’. The indications for MSCs include, but are not limited to inflammatory diseases, tissue repair and protection, neuronal disorder and, cellular vehicle for drugs and RNA. Thus far, there is no major report of safety issue with MSC transplantation. The seminar will show the efficiency of MSCs to deliver non-coding miRNA in the treatment of breast cancer and then discuss the issue of MSCs crossing the blood brain barrier. The question is whether MSCs may have a memory for homing and if tissue injury changes the homing properties of varied sources of MSCs.

The resistance to different therapies, especially chemotherapy is the major problem for treatment of most of the cancer including head and neck squamous cell carcinoma (HNSCC) and breast cancer (BC). Chemosensitivity of the tumours is associated with different molecular process like cell proliferation, apoptosis and competency of DNA damage response (DDR) activation. Among the different pathways associated with these molecular processes cell cycle, apoptosis, homologous recombination repair (HRR), mismatch repair (MMR) pathways are important due to presence of several cancer susceptible genes. To understand the association of these genes with chemo-tolerance, their expression were analysed in pre-therapeutic and neo-adjuvant chemotherapy treated (NACT) HNSCC and BC samples. The chemo-tolerant NACT HNSCC samples showed significantly low PCNA expression than the pre-therapeutic samples. However, significant high apoptosis index was observed in NACT HNSCC samples than pre-therapeutic. Comparatively increased expression of cell cycle negative regulators LIMD1 and RBSP3 and DDR associated MLH1 gene were seen in NACT HNSCC samples than pre-therapeutic, where as reduced expression of cell cycle positive regulators pRB and CMYC were evident in NACT HNSCC samples. Similar to NACT HNSCC, significantly low PCNA expression was seen in NACT BC than the pre-therapeutic samples. Low expression of CD44 was evident in NACT BC samples. However, comparable frequencies of deletion, methylation and overall alterations (deletion/methylation) of the DDR genes (BRCA1, BRCA2, FANCC, FANCD2, MLH1, MSH2) were found in both pre-therapeutic (29-78%) and NACT (32-76%) BC samples. The mRNA and protein expression of these genes showed concordance with the molecular alterations. Similar phenomenon has also been seen in BC cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin, where increased expression of the DDR response genes was evident. Quantitative methylation assay showed high frequency of hypomethylation of these genes in the BC cell lines after treatment of drugs as well as in the NACT BC samples. This has been seen to be due to reduce expression (mRNA/protein) of DNMT1 (DNA methyltransferase 1) in the samples. Thus, our data indicates that epigenetic modification of chromatin has important role in drug tolerance in cancer cell.

The role of epithelial-to-mesenchymal transition (EMT) has been documented in tumor invasion and metastasis of human breast cancer. However, the effect of ionizing radiation (IR) on EMT-like changes in human breast carcinoma cells has not been investigated in terms of mechanism of post-irradiation adhesion, migration and survival. The present study was conducted to investigate the IR-induced EMT and its underlying mechanism in MCF7 human breast carcinoma cells. Cancer cells were irradiated with single or fractionated doses of gamma-radiation (2-6 Gy). A dose-dependent change in cell morphology from cuboidal (cobblestone type) to an elongated spindle-like shape was observed. Compared to control, irradiated cells were observed with more filopodial/lamellipodial structures and formed less compact colonies. These changes seem to be associated with EMT as evidenced by downregulation of E-cadherin and upregulation of mesenchymal marker, vimentin. Irradiated cells (2-8 Gy/48 h) exhibited a significant increase in adhesion and migration abilities and stress fibre formation. Consistently, WB experiment showed that radiation, dose-dependently increased beta1-integrin and phosphorylated FAK and paxillin, which stabilize filamentous actin for intracellular stress fibres. These results suggested the involvement of integrin-FAK-paxillin signaling and actin remodeling in radiation-induced adhesion of MCF7 cells. Radiation was found to decrease alpha-/delta-catenins and Rho-GDI, a repressor of Rho-GTPases suggesting its role in the formation of filopodial/lammelipodial structures and actin stress fibre, which are required for cell adhesion and migration. Interestingly, our results revealed the dose-dependent upregulation of beta-catenin, a transcription factor, which transcribes genes for both EMT and survival/repopulation during cell migration. Taken together, our results revealed the intracellular signaling events, which seem to be responsible for post-irradiation survival and increased adhesion and migration of breast cancer cells. The possible implication of this work could be that the targeting of beta-catenin signaling during radiotherapy may prevent post-irradiation tumor recurrence and invasion/metastases.

APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. APE1 exists in complex with many cellular factors and raised the question how it is being recruited to execute a particular function. We recently showed that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, the loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB on to the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. In a more recent study, our collaborator documented that CUX1, which resides on 7q22.1, the most frequently and highly amplified chromosomal region in glioblastomas, caused resistance of glioblastoma cells to the mono-alkylating agent temozolomide, which damages the DNA by creatingapurinic/apyrimidinic (AP) sites. Further analyses revealed that CUX1 has the ability to stimulate APE1 AP endonuclease DNA repair activity that processes AP sites. Evidence will be provided showing that CUX1 exerts temozolomide resistance to glioblastoma cells, in part, by stimulating APE1 to process temozolomide-, as well as radiation-induced DNA lesions. Collectively, our findings reinforce the notion that APE1 represents a key target to intercept cancer progression.

IL-16 (Abstract No.: 0092)

Inhibition of DNA Repair as a Strategy to Treat Cancer

Sathees C. Raghavan

Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India

E-mail: sathees@biochem.iisc.ernet.in

Repair of DNA breaks is critical for maintenance of genomic integrity. DNA double-strand breaks (DSBs) are the most deleterious types of DNA damage. Nonhomologous end joining (NHEJ) is the predominant DNA DSB repair pathway in higher eukaryotes. DNA Ligase IV is one of the most critical components of NHEJ, involved in final sealing of DSBs. Inhibition of DSB repair pathway proteins can be used as a strategy to induce apoptosis in cancer cells. Recently we have chemically synthesized and characterized a novel inhibitor of Ligase IV, SCR7. Using radioactively labeled oligomeric substrates mimicking various in vivo DSBs, we showed that addition of SCR7 to rat testicular extracts abolished joining by NHEJ. Further, SCR7 interfered with the joining of compatible DSB ends catalysed by purified Ligase IV. Electrophoretic mobility shift and circular dichroism studies suggest that SCR7 binds to Ligase IV and interferes with its interaction to DNA ends. Further using animal models, we find that SCR7 treatment can inhibit progression of breast adenocarcinoma but not haematopoeitic cancers, resulting in a significant increase in life span. Interestingly, SCR7 impedes tumor progression in haematological cancers significantly, when co-administered with existing DSB inducing therapeutic modalities. More importantly, we show that when co-administered, SCR7 could reduce the effective dosage of γ-radiation from 2 Gy to 0.5 Gy, in cancers derived from breast cancer, colon cancer and B-ALL. Histopathological and immunofluorescence evaluation of tumor and other tissues suggest that the cytotoxicity induced is mostly restricted to the tumor. Besides, we observed that SCR7 can exist in multiple forms, although exhibited similar properties with varying efficiency. We also find that encapsulation of SCR7 in micelles can improve its efficacy by ~4-fold. Thus, by using various biochemical and biophysical approaches, we show that SCR7 is a potent inhibitor of NHEJ, can be used as a chemotherapeutic agent against multiple cancers.

IL-17 (Abstract No.: 0008)

Exosomes and Their Role in the Biological Response to Ionizing Radiation

Both directly irradiated and bystander cells can communicate each other using gap junction intercellular communication or by releasing exosomes into surrounding environment. Exosomes are well known to mediate communication between cells. Radiation-damaged tumor cells results in the activation of antigen presenting cells through the release of damage-associated molecular patterns (DAMPs). The main aim of this study is to identify the communication between radiated tumor cells and immune cells using exosomes. B16F10 GP melanoma cells were irradiated using 10 Gy or not using γ-rays produced from cobalt 60 unit. After irradiation, the cells were harvested for conditioned media and exosomes were isolated from it. Exosomes were characterized using Transmission electron microscopy (TEM), Immunoblot and Nanosight. DAMPs were identified in irradiated melanoma cells exosomes using Immunoblot method. To reveal the biological effectiveness of irradiated exosomes on immune cells, both radiated and non-irradiated melanoma cell exosomes were added to splenocytes. The immune cells differentially took up post radiation tumor exosomes than compared with non-irradiated tumor exosomes, suggesting that the content of exosomes can drive anti-tumor immunogenic process. These results indicated that there is an intriguing mechanism where exosomes could function to present radiation associated DAMPs to the immune system. To identify the other DAMPs within exosomes, exosome specific protein markers were screened and we found a significant difference between both radiation and non-irradiated cell exosomes. These results suggested that there is an impact in the production and biogenesis of exosomes after radiation treatment.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, 1Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Mumbai, Maharashtra, India

*Contributed equally

E-mail: shamiks@iitb.ac.in

Acquired radioresistance accompanied with increased metastatic potential is a major hurdle in effective radiotherapy of breast cancers. However, the nature of their inter-dependence and the underlying mechanism remains largely intangible. By employing radioresistant (RR) cell lines, we herein demonstrate that MCF-7 RR cells display phenotypic and molecular alterations evocative of epithelial to mesenchymal transition (EMT) with increased traction forces and membrane ruffling culminating in boosted invasiveness. We then show that these changes can be attributed to over expression of alpha-actinin-4 (ACTN4), with ACTN4 knockdown near-completely abrogating both radioresistance and EMT-associated changes. We further found that in MCF-7 RR cells, ACTN4 mediates the observed effects by activating AKT, and downstream AKT/GSK3β signalling. Though ACTN4 plays a similar role in mediating radioresistance and invasiveness in MDA-MB-231 RR cells, co-immunoprecipitation studies reveal that these changes are effected through increased association with AKT and not by overexpression of AKT. Taken together, our study identifies ACTN4/AKT/GSK3β as a novel pathway regulating radioresistance coupled invasion which can be further explored to improve the radiotherapeutic gain.

Non-targeted radiation effects (NTRE) refer the radiation effects observed in cells/tissues which have not directly received radiation, but manifest damages similar to irradiated cells/tissues. Such non-targeted effects of radiation are of two types, first, the effects to the close proximity/neighbouring cells (radiation induced bystander effect), second, effects at distant tissues/organs (abscopal effects). These effects are highly relevant in clinical outcome of cancer radiotherapy, but these have very limited knowledge in literature. Hence, to investigate in vivo NTRE under clinically relevant conditions, we have employed mouse (BALB/c) fibrosarcoma (WEHI 164 cells) tumor model. In the first approach, WEHI 164 cells, which were lethally gamma-irradiated (15 Gy), were implanted subcutaneously mixed with bystander cells. Irradiated tumor cells resulted in significant decrease in the growth of unirradiated (bystander) tumor cells. Histopathological analysis and other assays of these tumor samples showed decreased mitotic index, poor angiogenesis, increased apoptosis and senescence. Supernatant from irradiated cells showed changes in the level of cytokines and proteins favoring the mechanism of tumor growth inhibition. In partial tumor volume irradiation approach, a part of tumor was gamma-irradiated with a cone irradiator specially designed for teletherapy machine. Compared to whole tumor irradiation (which showed complete regression), partially irradiated tumors (~15% volume) showed ~60% reduction in tumor volume suggesting propagation of damaging bystander signals from irradiated to bystander cells. In another study, both hind legs of animals were transplanted with tumors, out of which tumor in one leg was irradiated (2 and 5 Gy) keeping another in shielded condition. Our results showed significant decrease in tumor growth in the shielded tumors. Taken together, our results provide deeper insight about NTRE in in vivo tumor model, which might have significant clinical implication under radiotherapy scenarios.

The radiotherapy treatment for cancer was initiated in early last century. The X-rays were invented in 1898 and its use for treatment for cancer initiated soon after. The knowledge of radiobiology for the tumor control and the side effects was very limited. Therefore, many patients suffered from the severe ill effects of radiation, however all these untoward effects have made possible to study deeply and newer techniques to improve the outcome of radiation therapy. Until 1980 the radiation treatment delivery equipment and methods were simple. The treatment plans were based on plain radiography and treatment plans were usually without tumor visualization. The treatments were associated with uncertainties and toxicities. Even the late toxicities may appear years after the completion of treatment and were associated with poor quality of life in many patients who survived for long time. Recent advances have improved the effectiveness, decreased in the complications, and also it translated in expansion of implications of radiotherapy. These advances include 3D-Conformal Radiation Therapy (3D-CRT), Intensity Modulated Radiation Therapy (IMRT), Stereotactic Radiotherapy (SRT/SRS), Brachytherapy, and Radioimmunotherapy. Each of these modalities has refined the radiation targeting and reducing radiation dose to normal healthy tissues. With understanding of radiobiology of tumors, for selected tumors a stereotactic radiosurgery/therapy (SRS/SRT), Stereotactic Body Radiation Therapy (SBRT), and Stereotactic Ablative Body Radiation Therapy (SABR) can be delivered in small number of fractions thereby saving patients attendance to hospitals. Recently, there is great interest in Hadron therapy due to its appealing physical properties to treat cancers and substantial reduction in the radiation dose to nearby tissues. The advance in imaging technologies (CT scan, MR Scan, PET, and SPECT) and powerful and faster computers computation has played a vital role. The cancer treatment advances are phenomenal technological advances and dosimetric improvements. There have been very few clinical studies to prove the improved outcomes on randomized pattern. There is need to evaluate that newer technologies are translating into widening the therapeutic index.

Chromatin acts as a natural hindrance in DNA-damage recognition, repair and recovery. Histone and their variants and isoforms undergo differential post-translational modification(s) and regulate chromatin structure to facilitate DNA damage response. During the presentation we will discuss the importance of chromatin organization and histone modification(s) during IR-induced DNA damage response in specific cell cycle phases of human cancer cells. Our earlier data have shown that G1-phase specific decrease of H3 serine10 phosphorylation in response to DNA damage is coupled with gain of γH2AX, global deacetylation and chromatin compaction in repair phase of DDR. The reversible reduction of H3Ser10P is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. Contrary to G1 phase, the decrease of H3Ser10P is not dependent on DNA damage but decrease correlates with cycling of cells to G1 phase. The recovery of H3Ser10P is affected in irradiated G2/M cells with efficiently arresting cells in G2 and resulting in cell death. Furthermore, in G1 cells, blocking of H3Ser10P by MKP1 inhibition impairs DNA repair process and results in poor survival of cells. Interestingly, cell cycle phases have different radio-sensitivity, with mitosis being the most radiosensitive. Thus, the differential H3Ser10P levels influence cell survival post IR, so could histone post-translational modifications and its modifying enzymes be epigenetic determinants of cell cycle phase specific radio-resistance? Therefore, the study raises the possibility of combinatorial modulation of H3Ser10P and histone acetylation with specific inhibitors to target the radio-resistant G1-phase cancer cells and thus may serve as promising targets for cancer therapy.

IL-22 (Abstract No.: 0123)

RAS Inhibition and Radiation for Cancer Therapy: A Polypharmacological Approach

Icahn School of Medicine at Mount Sinai, 1New York Structural Biology Center, 2Albert Einstein College of Medicine, New York, USA

E-mail: ep.reddy@mssm.edu

Oncogenic activation of RAS genes via point mutations occur in more than 30% of all human cancers, and investigations aimed at understanding the biochemical and biological mechanisms that are critical for the function of these oncogenic proteins is at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play a critical role in oncogenic RAS function, inhibiting this interaction constitutes an attractive and important therapeutic approach. Rigosertib is a novel Styryl Benzyl Sulfone which is Phase III clinical trials for Myelodysplastic Syndrome (MDS). Our studies show that rigosertib inhibits RAS signaling by a unique mechanism which involves its interaction with the RBDs of several RAS effector proteins, including RAF, the PI3K family of proteins as well as RalGDS. This interaction has been found to block the interaction of RAS with its effector proteins leading to the inhibition of multiple RAS signaling pathways. Rigosertib has been shown to have anti-tumor activity, alone and in combination with other chemotherapeutic agents, including radiation and Cisplatin. Cisplatin is the preferred primary chemotherapy for cervical malignancies and is used in combination with radiotherapy for the treatment of this disease. In an attempt to test the activity of rigosertib in enhancing the cytotoxic effects of radiation therapy, we used the cervical cancer cell lines, HeLa and C33A, to test the effects of rigosertib and cisplatin. In vivo tumor growth studies demonstrated higher performance of rigosertib when compared to cisplatin, with 53% longer tumor growth delay. These studies demonstrate that rigosertib is more effective than cisplatin when combined with radiation and caused minimal toxicity. These data support the need for clinical trials with rigosertib in combination therapy for patients with cervical carcinoma.

IL-23 (Abstract No.: 0116)

Study on the Radio-Modulating Effects of a Progestin Steroid, Anthracenediones and Prochlorperazine in Cancer Radiotherapy

Cancer is the second leading cause of death globally, and is responsible for 8.8 million deaths in 2015 as per WHO reports. Radiation therapy (RT) continues to play an important role in the treatment of 80% cancer. This study is based on repurposing of drugs for radiosensitization of tumour cells in order to reduce the damage caused by radiotherapy to normal tissues. The K-Ras is the most frequently mutated isoform present in 22% of all tumours analysed. There is substantial evidence linking Ras proteins to radioresistance. Pre-clinical studies have shown that inhibitors of K-ras can reverse radioresistance in human tumour xenografts expressing the mutant Ras oncogene. In search of novel K-ras targets, in-silico studies resulted into twenty FDA approved drugs that can potentially bind with K-ras. Out of these Norethisterone - a progestin steroid, Pixantrone - anthracenedione analogue, and Procholorperazine an antipsychotic drug are screened in the current study. These compounds exhibited Radiation Enhancement Ratio greater than 1 in MDAMB231, MCF7 and A549 cells at 2 Gy as observed by clonogenecity assay. These molecules showed considerable G2 phase arrest till 24 h in irradiated cells. Norethisterone on MDAMB231 cells showed 68% survival upon treatment with 300 µM of drug till 72 h of treatment. Further studies are in progress to elucidate mechanism of radiosensitization by these molecules.

James L Winkle College of Pharmacy, University of Cincinnati, Cincinnati, Ohio 45220, 1Department of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA

E-mail: kotaginh@ucmail.uc.edu

Despite the introduction of novel therapies, more than 90% of deaths from cancer are due to metastases that are resistant to conventional therapies. The major limiting factors for the effective use of chemotherapies in treating cancer metastasis is development of acquired resistance and the serious side effects caused by these drugs. Therefore, a new strategy that increases treatment efficacy, prevents relapse, provides a cure and reduces off target toxicity is needed. Photoactivated therapy is an attractive modality because it provides dual-selectivity and high level of control in targeted tumor cytotoxicity through a combination of direct cytotoxicity and immune-stimulatory mechanisms. However, the limited penetration of external light has limited the technology to treatment of surface accessible lesions. We have recently pioneered and demonstrated the concept of using ultraviolet-blue weighted light emitted from radiopharmaceuticals, for photoactivated therapy of cancers with high spatiotemporal precision and selectivity. Co-localization of the radiopharmaceutical and a photoactive drug in cancer cells facilitates therapy. The radiopharmaceutical serves as a light source to activate the drug in the cancer cells, while also serving as an imaging agent to concurrently monitor treatment response. Selective ablation of the cancer cells is achieved without significant off-target toxicity to normal cells and tissues. Our treatment strategy has the potential to expand the use of phototherapy for treating previously inaccessible metastatic disease using clinically relevant radiopharmaceuticals.

IL-25 (Abstract No.: 0095)

Modulation of Antioxidant Status and Cell Signaling for Radioprotection by Naringenin: A Phytochemical from Citrus Fruits

Ashu Bhan Tiku

Radiation and Cancer Therapeutics Laboratory, School of Life sciences, Jawaharlal Nehru University, New Delhi, India

E-mail: abtiku@mail.jnu.ac.in

Radiotherapy, in spite of being among the frontier therapeutic treatments for cancer, is limited by two major factors, one side effects on normal tissues and second development of therapeutic resistance in cancer tissues. The exact molecular events in irradiated cells are still being deciphered. IR is known to initiate various signaling cascades, involved in important cellular process, governing cell growth, proliferation and apoptosis. It is very well known that ROS generated during radiation can act as a secondary messenger and hence play a key role in signal transduction processes. The activation or inhibition of these pathways in response to radiation governs the ultimate fate of the cell. Some traditionally popular medicinal plants have recently gained attention for their ability to modulate a number of signaling pathways that could affect the fate of the cells exposed to radiations. One such agent is naringenin, a phytochemical mainly present in citrus fruits and tomatoes, and is well known for its positive health effects not only in cancer prevention but also in non cancer diseases. Naringenin, is a nontoxic molecule with a median lethal dose of >5000 mg/kg body weight in mice. In vitro studies, have show that naringenin protects the HaCaT cells against the UV radiation-induced damage by promoting the DNA repair and induces melanin production in murine melanoma (B16−F10) cell lines. The potential of Naringenin as a radioprotective agent was evaluated in our lab in whole body irradiated mice against gamma radiation-induced DNA, chromosomal and membrane damage. Naringenin pretreatment was found to increase the antioxidant status of irradiated mice. Multiple factors operating at cellular and molecular levels led to an increased survival of mice exposed to sub lethal doses of gamma radiations. Although naringenin induces apoptosis in cancer cells we found that it can protect against radiation-induced apoptosis in normal cells and modulate NF-kB pathway. The results from the present study indicate that naringenin can afford radioprotection at the cellular, tissue and organism levels.

IL-26 (Abstract No.: 0013)

Radioimmunotherapy of B-Cell Lymphoma: Approach to Enhance Apoptotic Cell Death

Radioimmunotherapy (RIT) is a branch of nuclear medicine wherein antibodies are employed to deliver cytotoxic dose of beta/alpha radiation to the targeted sites. The promising therapeutic results of Bexxar® and Zevalin® in lymphoma provide reasonable anticipations for effectiveness of RIT. Benefit of RIT is not only limited to its particulate nature of radiation but it can also target and treat tumor metastases and effectively kill the radioresistant hypoxic cells. Lymphomas are a heterogeneous group of hematological malignancies, affecting mostly B-cells. There are several therapeutic modalities for B cell malignancies. Even though, rituximab is an immunotherapeutic agent used for treatment of NHL patients, only 50% patients showed clinical response. To enhance cell killing rituximab was tagged with beta emitting radionuclide and further expected to increase by pretreatment of chemotherapeutic drugs such as doxorubicin/camptothecin. Raji cells were pre-treated with drugs followed by incubation with 131I-rituximab and various parameters were assessed to study magnitude of apoptosis and lurking mechanism of cell death. Apoptotic cell death was highest in combination of camptothecin/doxorubicin compared to rituximab/131I-rituximab alone. Raji cells undergo G1 arrest upon 131I-rituximab treatment, which might be the cause of apoptosis, confirmed by downregulation of bclxl and cleavage of PARP. Expressions of p38 MAPK were decreased in combination treatment of camptothecin/doxorubicin and 131I-rituximab however, phosphorylation of p38 increased. These studies conclude that camptothecin/doxorubicin in combination with 131I-rituximab enhanced apoptotic cell death in B-cell lymphoma tumor cells which might be the efficacious treatment modality for the lymphoma patients in general.

Increased industrialization and population growth have led to the generation of huge amount of waste containing various kinds of toxic environmental pollutants (e.g., dyes, metal ions, pharmaceuticals, etc.), leading to various health issues. Radiation technology can offer viable solutions to some of these environmental problems. Radiation processing of polymers using ionizing radiation offers environment friendly, efficient and versatile tool for the bulk as well as surface modification of polymeric materials. Different types of functional polymeric materials were developed via radiation grafting of suitable monomers with epoxy and quaternary ammonium groups (EPMA, VBT, etc.) on to range of low cost polymer supports (PP, PES, Cellulose, etc.) and employed for environmental remediation. Different grafting parameters (dose, monomer concentration, ambient, etc.) were optimized to achieve the desired level of grafting extent. The functional polymers were characterized by different techniques, such as grafting yield estimation, elemental analysis, TGA, FTIR, SEM, etc. Two strategies used for environmental application of these functional polymers include the fabrication of (i) Enzymes (Laccase) and metal nanoparticles (Pd, Au)-immobilized polymer based recyclable catalytic system for remediation of toxic pollutants (Dyes, Cr (VI)) from waste water and (ii) Low cost, efficient and recyclable adsorbents functional polymer adsorbents for removal of toxic pollutants (dyes and metal ions) from waste water systems in batch as well as continuous flow mode. Enzyme-immobilized catalytic system was repeatedly used for catalytic degradation of textile dyes, where as metal (Pd) immobilized system was used for reduction of Cr (VI) to less toxic Cr (III) in batch as well column mode.

IL-28 (Abstract No.: 0119)

A Preliminary Study to Assess the Impact of Stem Cell Sparing Radiation Therapy on the Outcomes of the Treatment of Glioblastoma Multiformae

Radiation therapy with Temozolamide has emerged as a standard of care in the treatment of Glioblastoma Multiformae. Radiation therapy has the potential of being cytotoxic despite blood brain barrier and Temolozamide an alkylating agent is one of the few cytotoxic drugs with the potential to cross blood brain barrier. Radical radiation therapy of GBM carries a risk of inducing cognitive decline. It is assumed that sparing neural stem cells scattered discretely in niche areas in Sub Ventricular Zone (SVZ), Subgrannular zone (SVZ) and Hippocampus would promote repair, which in turn may reduce the possibility of cognitive decline. Contrary to this assumption there seems to be a valid argument for not sparing above zones as these niches may harbour cancer stem cells which migrate to tumour and create a nidus. The current study was to study the impact of sparing neural stem cells. The assumption is that sparing neural stem cells will not adversely affect survival as has been assumed by certain groups. Patients with histologically proven Glioblastoma Multiformae underwent chemo-radiation with intensity modulated radiation therapy with stem cell sparing or with three-dimensional conformal radiation therapy. All patients were scheduled to receive a total dose of 60 Gy in 6 wks and 30 fractions. Oral temozolamide was administered daily for all six weeks at a dose of 100 to 140 mgs was administered along with radiation. Stem cell niches were contoured in all patients though dose constraints were assigned to stem cell sparing group only. Patients were planned on Monaco Treatment Planning System (V 5.11.02) and treated with 6MV. linear accelerator. During treatment patients were analysed retrospectively. A total of 46 patients were evaluated. One patient in IMRT arm and two in 3DCRT were censored from the study for not having received 60 Gy. The average survival in both groups was 14.4 and 14.6 months. The chi square test is 1.67 and the chi-square 2=0.1960. Hence, the hypothesis that survival from the two treatments does not significantly differ cannot be required. GBM are a heterogeneous group of tumours with a tendency to be aggressive, Limetal and chenetal have demonstrated a higher incidence of failure in those patients where it was in close proximity of niche areas of stem cell. This is presumed to be due to migration of stem cell like tumour cells to different areas including tumour. In fact neural stem cells share many of the phenotypical characters of cancer stem cells though it is extremely difficult to distinguish between neural and cancer stem cells. The range of sensitivity of stem cells to radiation is not clear. The available literature suggests a decline in neuro-cognitive decline to a dose as low as low as 7 Gy. It has been suggested that neural stem cells lodged in hippocampus and other niche areas die at a low despite being sensitive. The dose escalation studies in the treatment of GBM haven’t shown any improved survival. Dose escalation inevitably increases the dose to NSC. Thus it stands to reason that sparing NSC will not reduce the probability of better outcome following radiation. The current retrospective study did not show any difference in survival though NSC received in IMRT group received much less radiation than in 3CDRT group. A large cohort with multiple dose levels should help clarify the dose response outcomes following stem cell sparing techniques.

Departments of 1Cell Biology and 2Radiation Oncology, University of Groningen, University Medical Centrum Groningen, Groningen, The Netherlands

E-mail: r.p.coppes01@umcg.nl

The response of normal tissues to irradiation is mainly determined by the survival and regenerative potential of the tissue stem cells, and modulated by inflammatory processes, vasculature damage and altered neuronal innervation and fibrosis. Interestingly, transplantation of tissue specific stem cells has been shown to restores tissue homeostasis and prevent late radiation effects. Moreover, the sparing of localized stem cells was predicted to preserve salivary gland function in patients treated for head and neck cancer. Interestingly, mounting evidence indicates that cancer stem cells might contribute to the poor prospects. Recently, we and others have developed methods to culture patient specific organ and tumour stem cell containing organoids (tissue resembling structures). These organoids contain all the tissue/tumor lineages and the tissue/tumor stem cells, as indicated by their secondary organoids self-renewal potential and regeneration/regrowth potential and offer the opportunity to investigate tissue and patient specific assessment of the response of stem cells to (chemo-) radiotherapy. Stem cell survival curves and DNA DSB repair kinetics indicate that the response of organoids to different radiation qualities may differ from tissue to tissue, especially in the low dose regions typically delivered to the normal tissue outside the planning target volume. Therefore, organoids cultures could be used to investigate the mechanism of differences in response of normal and tumour stem cells to irradiation and exploit these for personalized optimisation of (chemo-) radiation treatment and prediction of treatment response.

Stem cells have the ability to self-renew over the lifetime of an organism and also to differentiate into multiple cell lineages. The majority of mammalian cells in situ originate from a corresponding progenitor daughter cell that is terminally differentiated. Various factors, including reactive oxygen species (ROS) that accumulate during differentiation and over the stem cell lifespan can cause DNA damage. In addition, differentiation-dependent changes in chromatin structure and transcriptional alterations can also impact genomic integrity by altering the DNA damage response (DDR) and repair facility. Thus, genomic stability is likely to be under increased stress during differentiation. How factors that induce differentiation, like NO donors, impact stem cell genomic stability is unclear. Since nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase specific chromosomal aberration after exposure to DNA damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on DSB repair by NHEJ but reduced DSB repair by HR. Our studies suggest that DNA repair by HR is impaired in differentiated cells.

IL-31 (Abstract No.: 0023)

Effects of Low Doses of Radiation on Human Cells: Time for Fresh Perspective

Current radiation protection policies are based largely on extrapolation from effects at high doses and assume that the excess risk from low level exposure is directly proportional to dose (Linear-No-Threshold hypothesis, LNT). These presumptions have fueled unjustified fear of radiation in public and scientific community alike. Ongoing studies from our lab on High Level Natural Background Radiation Areas (HNLRA) of Kerala, India, integrating biological and epidemiological studies, have challenged this hypothesis. A quantitative proteomic approach to assess responses to low dose radiation exposures identified 1570 proteins with statistically significant (p≤0.05) changes in abundance (>1.3 fold) between individuals from HLNRA as compared to individuals from Normal Level Natural Background Radiation Areas (NLNRA). Almost 215 biological processes were found to be differentially modulated in HLNRA samples. The top biological pathways identified using bioinformatics included stress activated protein kinases and many DNA repair networks. The data suggested that a subset of the proteins and signaling pathways are differentially regulated at low and high doses. Thus, extrapolating risk linearly from high to low doses may not accurately assess the actual risks of low-dose exposures. In addition, our data on adaptive responses in human lymphocytes, using either in vitro irradiated human lymphocytes or samples from HLNRA, indicated decreased damage and increased protective pro-survival mechanisms with low doses. This puts into question some of the established radiobiological paradigms and warrants caution in interpretation of estimated risks using LNT model.

IL-32 (Abstract No.: 0057)

Enhanced Biomarkers after Medical Radiation Exposure: Will They Increases Risk of Stochastic Effects?

Medical applications of radiations are immense: either diagnosis or therapy. Owing to the significant advantages it is preferred choice then other modalities. Nevertheless, its usage is not devoid of concerns; it was attributed due to an altered structure of proteins and nucleic acids, generally known as biomarkers of radiation exposure. Presence of evidences (in-vitro and in-vivo) on the relationship between high dose and high dose-rate induced biomarkers and the stochastic effects, resulting into the strong belief that even low dose and dose rate radiation induced biomarkers can increase such risk in exposed individuals. For an example, the radiation specific biomarkers “dicentric chromosomes” (DC) can’t be discriminated that induced by low dose radiation exposure from that of high dose radiation exposure. Is it appropriate to use the marker of exposure (DC) to investigate its effects? We have measured a range of biomarkers (DC), micronuclei (MN), translocation (TL), gammaH2AX (γ-H2AX), Comet assay, P53ser15 expression in blood samples and normal as well as tumor cells exposed in-vitro and subjects exposed during therapy, imaging and occupational radiation workers. The frequency of CA in interventional personals (0.101 ± 0.001) is higher than healthy volunteers (0.0004±0.0004); in patients underwent CT, the frequency is higher (0.009 ± 0.01) than before taken the CT (0.0004 ± 0.0004). A clear dose dependent increase in the level of those markers was observed among cell lines followed by in-vitro exposure to ionizing radiations. Moreover, both tumor and normal cells irradiated to therapeutic range of doses (2 and 4 Gy), showed a non-targeted effects (bystander response and genomic instability); however, exposure of cells to diagnostic range (~250 mGy) failed to induce those effects. Even, frequency of stable aberrations (TL) measured in occupational health workers did not show any increase. Thus our results suggest that low dose occupational exposure induced biomarkers are not able to predict the risk effects accurately.

IL-33 (Abstract No.: 0066)

Radioactive Material Contamination in Food after the Fukushima Nuclear Power Station accident

T. Aono, M. Akashi1, M. Fukuda, S. Yamazaki, S. K. Sahoo

National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 1National Institutes for Quantum and Radiological Science and Technology, Chiba 263-8555, Japan

E-mail: aono.tatsuo@qst.go.jp

A nuclear accident of the Fukushima Daiichi Nuclear Power Station (FDNPS) had been caused by the tsunami after the Great East Japan Earthquake in March 2011, and the huge amounts of radionuclides were dispersed around Fukushima immediately. It is important that monitoring of activity concentrations of radionuclide in the territorial and marine environment for understanding the dispersion and behavior of radionuclide after the FDNPS accident. This study aimed to examine the temporal and spatial variations of activity concentrations of radionuclide in marine environment and to discuss the effects of radioactive material on products as food after the FDNPS accidents. After this accident, Nuclear Emergency Response Headquarters in Japan started the use of the provisional regulation value in food promptly (March 17, 2011-March, 2012). This concept of the limit for food and water is less than 5 mSv/yr. However, it was necessary to carefully consider external and internal exposure. Based on the concept of 1 mSv/yr for food and drinking water, new standard value in food (the limit of general food: 100 Bq-Cs/kg) was enforced from April 1, 2012. The concentrations of radiocaesium (radio-Cs) in seawater are almost same levels before the accidents except the near site of FDNPS, and these in fish generally decreased, and radiation doses from foods intake are negligible. However, high activity concentrations of Cs are kept to some specific products as food as the result of nutrient cycle, accumulation, translocation and food chain. In this presentation, we would like to talk about the monitoring, distribution and behavior of radio-Cs in marine environment before and soon after the FDNPS, the estimation of dose rate in marine biota around Fukushima, the monitored range and trend of radio-Cs in food and estimation of annual effective dose from radio-Cs in food.

IL-34 (Abstract No.: 0111)

Internal Dosimetry: Challenges in Biokinetic Modelling and Decorporation of Radionuclides

Radiation exposure, both external and internal, may occur during the operation of various nuclear fuel cycle facilities and radiation facilities. Internal dose is the radiation exposure that results from the intake of radioactive materials into the body by inhalation, ingestion, absorption through the skin or via wounds. Assessment of internal doses can be divided into two phases, namely determination of the amount of radioactive material in the human body, in body organs or wounds by direct measurements and/or by indirect methods like excretion analysis or air monitoring followed by interpretation of the monitored data in terms of intake and/or internal dose. Determination of internal doses is a complex procedure that requires the use of biokinetic and dosimetric models which describe the behavior of the radionuclides and the deposition of their energy in the tissues. In the present talk, ICRP models will be introduced along with the need for development of similar models for Indian population. Effective and prompt medical countermeasures for decorporation of actinides in case of radiological/nuclear emergencies, i.e. mass contamination scenarios Challenges faced in development of biokinetic models for Indians are discussed. Decorporation treatment is most effective when administered shortly after contamination before the radionuclides become fixed in tissues. Current decorporation treatment options especially for actinides do not meet the challenge imposed by a mass casualty setting. An overview of factors that affect the efficacy of decorporation treatment and recent developments in this field are also covered in the present talk.

IL-35 (Abstract No.: 0054)

Radiocaesium and Radiostrontium Distribution around Fukushima (Japan) after Nuclear Power Plant Accident

S. K. Sahoo, N. Kavasi, S. Kasar, H. Arae, T. Aono

National Institutes for Quantum and Radiological Science and Technology, National Institute of Radiological Sciences, Chiba 263-8555, Japan

E-mail: sahoo.sarata@qst.go.jp

The nuclear accident at the Fukushima Daiichi Nuclear Power Plant (FDNPP) triggered by earthquake caused tsunami in March 2011, with massive contamination of radionuclides in Japan. In the environment, radiocesium isotopes (134,137Cs) and 90Sr have presence due to their long-lived half-life. Long-lived radionuclides deposited on soil can cause an enhanced radiation exposure even after many years and depending upon environmental conditions can be mobilized to aquatic systems. Therefore, the assessment of the fate and transfer of these radionuclides in the soil-water system is very important for radiation protection and dose assessment. Study of the vertical migration process of radiocesium is important to evaluate the external irradiation from gamma-emitters in soil and to estimate the plant root uptake. With the understanding of the migration process downwards into the soil it is possible to establish a vertical profile as a function of time. High Cs activity was observed in the areas in the plume direction during the accident. Strontium as an alkaline earth metal shows similar chemical behavior to calcium, thus it can be accumulated by bone tissue causing internal radiation exposure. Therefore the environmental monitoring of 90Sr is another important tasks in Japan after the Fukushima accident. The 90Sr (T1/2 28.8 y) is a man-made radionuclide so-called the fission product of nuclear reaction (235U (n,f)90Sr) and released into the environment by nuclear weapon tests, nuclear facilities (accident or normal operation), reprocessing plants. Due to the atmospheric nuclear weapon tests, the soil in Japan is contaminated with 90Sr. To understand the geochemical behavior of Cs and Sr, distribution coefficient (Kd) and soil parameters have been determined experimentally and the important parameters affecting Cs and Sr sorption have been identified. The present study will be useful for understanding the migration processes and for long-term prediction of activity depth profiles in soil.

Galactic cosmic radiation presents a major challenge to human exploration and long-term residence in space. While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no source duplicates the unique space radiation environment. We are developing a biosensor-based nano satellite planned to launch in 2019 as a secondary payload aboard NASA’s Space Launch System first Exploration Mission (EM-1), from which it will be deployed on a lunar fly-by trajectory and into a heliocentric orbit. Our biosensor uses the yeast Saccharomyces cerevisiae to measure the yeast’s DNA damage response to ambient space radiation, which will be compared to information provided by an on-board radiation dosimeter and to data obtained in Low Earth Orbit on the International Space Station, and on Earth. Desiccated cells will be carried within microfluidic cards, and each card will be activated by medium addition at different time points during the mission. Cell growth and metabolic activity will be tracked continuously via optical density. One reserve set will be activated if a solar particle event occurs during the mission. NASA’s BioSentinel mission will conduct the first study of biological response to space radiation outside Low Earth Orbit in over four decades. BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments. BioSentinel’s results will be important for improving interpretation of the effects of space radiation exposure, and for reducing the risk associated with long-term space exploration.

Lepidopteran insect cells are an intriguing model of radiation response since these can display over 100 times higher radioresistance than mammalian cells despite carrying numerous homologies. Extensive studies are conducted in our laboratory for understanding the cellular responses and molecular pathways induced by radiation/stress in these cells. Cellular radiation/stress responses of Sf9 ovarian cell line derived from Spodopterafrugiperda, the Fall armyworm (order Lepidoptera; class Insecta) were studied. Sf9 cells display excessive resistance to gamma-radiation doses up to 1000 Gy, with the iso-effect doses inducing DNA damage varying by 10 times. Cell death was observed primarily at doses >1000 Gy, about 100 times higher than radiation doses lethal for human cells. Mitochondrial/calcium disturbances were detected only at death-inducing high doses, leading to typical apoptosis. Mitochondrial pathway of apoptosis was also induced by actinomycin-D with excessive sensitivity in Sf9 cells, and cytosolic cytochrome-c release was Bax-mediated/mPTP-independent in nature. These cells have significantly stronger antioxidant mechanisms accompanied by a prominent absence of NOS-induced NO-mediated response. Proteomic profiles indicated a stronger mitochondrial response with alterations in expression of much lesser number of proteins following irradiation. Peculiar differences in chromatin organization also prevent DNA damage and enhance DNA repair, with interesting roles played by core histones. These cells also showed dependence on certain micro-RNAs known to regulate cell death in Drosophila, indicating presence of alternate/additional mechanisms. We have also discovered a ‘p53-dependent gateway’ regulating radiation-induced cell death through suppressing miR-31 overexpression. As a result, Lepidopteran cells can dodge radiation-induced cell death quite effectively.

IL-38 (Abstract No.: 0010)

Ultraviolet Radiation and the Skin: Photobiology and Natural Photoprotectors

Ultraviolet radiations (UVR) have more energy than visible light but not as much as the ionizing X-radiation. The shorter waveband of solar UVR (UVB; 280–320 nm) mainly affects the epidermal layer of the skin and develops non-melanoma type skin cancers. Whereas, the longer wavebands of solar UVR (UVA; 320–400 nm) penetrate deeper into the dermis through the epidermis induces solar elastosis, inflammatory signaling and photoaging. We systematically studied cellular and molecular changes caused by both UVA and UVB wavebands using suitable experimental models. We observed even very low single UVA exposure (10 mJ/cm2) generates reactive oxygen species (ROS); elicit oxidative DNA damage and apoptosis in human skin epidermal cells (HaCaT). A short-term whole body UVA irradiation (a sum of 100 J/cm2 in 10 consecutive days) into the mouse skin visibly induces wrinkles and sunburn which have been linked with the overexpression of inflammatory proteins. This UVA induced photoaging events were significantly reverted by a lipophilic skin permeable monoterpenoid α-pinene (AP). Moreover, AP inhibited expression of matrix metalloproteinases (MMP-1 and MMP9) and collagen IV degradation in this short-term UVA-exposed mouse skin. Whereas, exposure to UVB wavebands generates entirely a different spectrum of biological effects in the skin cells. Exposure to UVB radiation-induced significant CPDs formation in human skin dermal HDFa cells and the frequencies of CPDs were reduced by a dietary flavonoid apigenin (15 μM). Further, this phytochemical restored UVB-induced loss of NER related proteins in skin dermal derived HDFa cells. In another study, we exposed mouse skin to 180 mJ/cm2 of UVB thrice weekly for 30 weeks. This chronic UVB exposure caused skin tumor which has been confirmed as squamous cell carcinoma (SCC) and this tumor incidence was linked with the upregulation of carcinogenic signaling in the mice skin. Treatment with a proven chemo-preventive phytochemical, caffeic acid (CA), before each UVB exposure significantly reduced tumors multiplicity through modulating signal transduction pathways in the 30 weeks UVB exposed mouse skin. Various cellular and molecular events developed during different wavebands of UVR exposure in the different parts skin layers and the prevention strategies using suitable countermeasures from natural origin will be discussed in this presentation.

Living organisms protect themselves from continuous exposure to naturally occurring radiation in environment, radioactive elements in their tissues etc. Growing concern about effect of radiation on human health and natural ecosystems are those exposures from manmade sources. Deinococcus radiodurans a gram positive extremophile bacterium is an ideal prokaryotic model for studying tolerance for very high doses of gamma radiation stress, and the invertebrate model organism Chironomus ramosus established by us, is suitable for studying effect of low to moderate doses of gamma radiation in eukaryotes. Compared to humans, D. radiodurans is 1000 times and C. ramosus is 350 times more tolerant to gamma radiation. Present study was undertaken to look for non coding and coding transcripts that are expressed during post irradiation recovery (PIR) period in these 2 primitive organisms. This would address the molecular interplay associated with their respective radio-tolerance mechanism. An open source RNA analysis pipeline – ‘READemption’ was used for high throughput sequencing (HTS) data analysis. Non coding RNAs expressed within operons of D. radiodurans were previously unreported and few of these were unique to the species. In C. ramosus, 90 genes were differentially expressed during 04 h PIR and 233 showed >4 fold expression (P<0.05) after 24 h PIR. Genes involved in nucleotide binding, iron ion binding, catalysis, DNA damage and repair, potassium channel, transporters etc. were common to both organism. Importance of genome wise HTS for studying evolution of common molecular processes involved in radio-tolerance in prokaryote and eukaryote model system could be established by this study.